This proposal addresses the origins of the vertebrate adaptive immune system, specifically by revealing the regulatory mechanisms of a recently discovered class of immunological genes. How is the lamprey variable lymphocyte receptor gene regulated? Also, is it regulated by the transcription factors homologous to those which direct mammalian lymphocyte geneexpression? A combination of bioinformatic and molecular approaches will be used to define the cis- and trans- regulatory elements of the variable lymphocyte receptor in the agnathans. Comparative sequenceanalyses and cis-regulatory module predictions will be performed, and if inadequate, new methods will be developed. EMSAs will test for the binding of transcription factors to regulatory motifs. Transactivation analyses will determine if specific transcription factor- cis-regulatory module combinations can regulate transcription. These experiments should answer the questions above and elucidate the origins of the adaptive immune system.